Fluorescein-labeled ThUBD probe for super-sensitive visualization of polyubiquitination signal in situ cells

Huang, Shuai; Gao, Yuan; Wang, Yonghong; Li, Fengzhi; Xiao, Weidi; Ge, Woo-Ping; Li, Yanchang; Xu, Ping

doi:10.1016/j.talanta.2022.123564

Cited by 2 publications

(3 citation statements)

References 33 publications

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“…We first compared the ubiquitination levels of the WT and ubp7 Δ strains. Equal amounts of WT and ubp7 Δ TCL were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with ThUBD-HRP ,− to detect the polyubiquitination signal. As expected, the results showed that ubp7 Δ had a substantially higher ubiquitination signal, nearly double that of the WT (Figure A,B), which was also consistent with the findings from LC–MS/MS analysis (Figure C).…”

Section: Resultsmentioning

confidence: 99%

“…To systematically investigate the substrates of Ubp7 based on the ubiquitomics quantification, the WT and ubp7 Δ strains were labeled with SILAC medium, and then the UbC samples were enriched with Ni-NTA resin or ThUBD ,− (Figure A). The enriched UbC was separated by SDS-PAGE, and each gel band was cut into eight slices based on the molecular weight (Figure B).…”

Section: Resultsmentioning

confidence: 99%

“…TCL and UbC were resolved on 10% SDS-PAGE, transferred onto the nitrocellulose membranes (Bio-Rad, Hercules, CA), and then detected by standard immunoblot analysis. The antibodies used for this study included ThUBD-HRP, ,− anti-Ub-K48 antibody (EMD Millipore, cat. no.…”

Section: Methodsmentioning

confidence: 99%

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Quantitative Proteomics-Based Substrate Screening Revealed Cyclophilin Stabilization Regulated by Deubiquitinase Ubp7

et al. 2023

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Quantitative proteomics has emerged as a crucial approach to identifying ubiquitinated substrates to investigate the functions of ubiquitination in cells. In this regard, although the substrate screening of certain enzymes in the ubiquitin system has been based on proteome or ubiquitinome level measurements, the direct comparison of these two approaches has not been determined to date. To quantitatively compare the efficiency and effectiveness of substrate screening from the entire proteomics to the ubiquitinomics filter, we used yeast deubiquitinating enzyme, Ubp7, as an example to evaluate it in this study. A total of 112 potential ubiquitinated substrates were identified from the ubiquitinomics level, whereas only 27 regulated substrates were identified from the entire proteomic screening, demonstrating the increased efficiency of ubiquitinomics quantitative analysis. Subsequently, we selected cyclophilin A (Cpr1) protein as an example, which was filtered out at the proteomics level but was a promising candidate according to the ubiquitinomics filter. Additional investigations revealed that Cpr1 possessed a K48linked ubiquitin chain regulated by Ubp7, which may affect its homeostasis and, consequently, sensitivity to the therapeutic drug cyclosporine (CsA).

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